Tim 3 apc cy7

Manufactured by BioLegend

TIM-3 (APC-Cy7) is a fluorescence-labeled antibody that binds to the T-cell immunoglobulin and mucin domain-containing protein 3 (TIM-3). TIM-3 is a cell surface protein expressed on various immune cells, including T cells, natural killer cells, and dendritic cells. The APC-Cy7 fluorochrome is used to label the TIM-3 antibody, allowing for the detection and analysis of TIM-3-positive cells using flow cytometry.

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Characterization of CAR-T Cells by Flow Cytometry

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The following antibodies were used: CD3 (VioBlue; Miltenyi Biotech or Pacific blue and PE; BioLegend), CD4 (FITC or APC-Cy7; BioLegend), CD8 (PE-Cy7; BioLegend), CD3/CD19 antibodies (FITC/PE; BD), CD28 (PerCP-Cy5.5; eBioscience), PD-1 (FITC; clone: EH12.2H7; BioLegend), TIM-3 (APC-Cy7; BioLegend), LAG-3 (VioBlue; Miltenyi Biotech), CD45RA (APC-Vio770; Miltenyi Biotec or Brilliant Violet; BioLegend), CCR7 (PerCP-Vio770; Miltenyi Biotec or PerCP; BioLegend), CCR2 (APC; Biolegend), CCR4 (PE; Biolegend), CCR5 (Alexa Influenza 488; Biolegend), CXCR2 (PE-Cy7; Biolegend) and CXCR3 (FITC; Biolegend).
Transduction efficacy was determined on day 6 and day 9 of culture by labeling CAR-T cells with biotin-labeled polyclonal goat anti-mouse F(ab)₂ antibody (anti-Fab, Jackson Immunoresearch, West Grove, Pennsylvania) and streptavidin (APC conjugated; BioLegend). CD3+F(ab)₂+ cells were defined as CAR-T cells. Isotype labeled cells (Jackson Immunoresearch) and untransduced cells served as negative controls. For further characterization, cells were stained with antibodies mentioned above. Cells were washed and re-suspended in cell staining buffer (BioLegend), incubated for 30 min with the antibodies on ice, washed and measured using MACSQuant FACS cytometer (Miltenyi Biotec). Samples were analyzed by FlowJo software (FlowJo LLC, Ashland, Oregon).

Itzhaki O., Jacoby E., Nissani A., Levi M., Nagler A., Kubi A., Brezinger K., Brayer H., Zeltzer L.A., Rozenbaum M., Vernitsky H., Markel G., Toren A., Avigdor A., Schachter J, & Besser M.J. (2020). Head-to-head comparison of in-house produced CD19 CAR-T cell in ALL and NHL patients. Journal for Immunotherapy of Cancer, 8(1), e000148.

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CAR T Cell Phenotypic Characterization

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Eight days after activation, CAR T cells were either maintained alone or cocultured with SKBR3 tumor cells for 8 and 24 h under control and pro-oxidative conditions. Then, CAR T cells were collected, fixed, and stained to assess: i) CAR construct expression, ii) exhaustion state, and iii) memory state. The following antibodies were used for staining: i) CAR construct expression, IgG-PE (Southern Biotech, 2042-09); ii) exhaustion state CD3-PE (BD Biosciences, 345765), CD4-V450 (BD Biosciences Biosciences, 560345), CD8-FITC (BD Biosciences, 555366), PD1-APC (BioLegend, 367406), TIM3-APC-Cy7 (BioLegend, 345026), and LAG3-PE-Cy7 (BioLegend, 369310); and iii) memory state CD4-AF700 (BioLegend, 317426), CD8-APC-Cy7 (BioLegend, 344714), CD45RA-APC (BioLegend, 304112), CD45RO-PerCP-Cy5.5 (BioLegend, 304222), CCR7-PE-Cy7 (BioLegend, G043H7), CD62L-PE-Cy5 (BioLegend, 304808), Granzyme B-FITC (BD, 558132), IFN-γ-BV621 (BioLegend, 502536), CD127-BV421(BioLegend, 351310), and CD57-PE (BioLegend, 359612) according to the manufacturer’s instructions.

Balta E., Janzen N., Kirchgessner H., Toufaki V., Orlik C., Liang J., Lairikyengbam D., Abken H., Niesler B., Müller-Decker K., Ruppert T, & Samstag Y. (2022). Expression of TRX1 optimizes the antitumor functions of human CAR T cells and confers resistance to a pro-oxidative tumor microenvironment. Frontiers in Immunology, 13, 1063313.

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